Calcium phosphopeptide complexes

ABSTRACT

Phosphopeptides containing the Ser(P) cluster sequence motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu- can stabilize their own weight in amorphous calcium phosphate (ACP) and amorphous calcium fluoride phosphate (ACFP). The amorphous phases stabilized by the phosphopeptides are an excellent delivery vehicle to co-localise calcium, fluoride, and phosphate ions at the tooth surface in a slow-release amorphous form producing superior anticaries efficacy. These amorphous phases stabilized by the phosphopeptides also have utility as dietary supplements to increase calcium bioavailability and to help prevent diseases associated with calcium deficiencies.

The present invention relates to novel complexes in which amorphouscalcium phosphates are stabilised by phosphopeptides. These complexeshave anti-cariogenic effects, and may also be used as dietarysupplements to increase calcium bioavailability and to heal or preventdiseases associated with calcium deficiencies. Methods of making thecomplexes of the invention and of treatment or prevention of dentalcaries, calcium malabsorption, and bone diseases are also provided

BACKGROUND Dental Caries

Dental caries is initiated by the demineralisation of hard tissue on theteeth by organic acids produced from fermentation of dietary sugar bydental plaque odontopathogenic bacteria

Even though the prevalence of dental caries has deceased through the useof fluoride in most developed countries, the disease =as a major publichealth problem. The estimated economic burden of treating dental cariesin Australia in 1991 was $471 million, being higher an that for otherdiet-related diseases including coronary heart disease, hypertension orstroke.

In developing countries where the availability of industrialised foodproducts is increasing, prevalence of dental caries is also increasing.Recent studies have highlighted a number of socio-demographic variablesassociated with the risk of developing caries; high risk is associatedwith ethnicity and low socio-economic status. The level of high-riskindividuals has remained constant even though the overall severity andprevalence of disease in the community has decreased. Dental caries istherefore, still a major public health problem, particularly in ethnicand lower socioeconomic groups. This highlights the need for anon-toxic, anticariogenic agent that could supplement the effects offluoride to fit lower the incidence of dental caries. An agent whichwould reduce the dose of fluoride required to reduce the incidence ofcaries would be particularly desirable in view of community anxietyabout fluoride, and in view of the fact that fluorosis can develop evenat currently used doses.

The food group most recognised as exhibiting anticaries activity isdairy products (milk, milk concentrates, powders and cheeses). U.S. Pat.No. 5,130,123 discloses the component responsible for thisanticariogenic activity as casein. However, the use of casein as ananticariogenic agent is precluded by adverse organoleptic properties andthe very high levels required for activity.

Preliminary investigations determined that tryptic caseinphosphopeptides contributed to the anticariogenic activity and this wasmade subject of U.S. Pat. No. 5,015,628. In particular, peptides Bosα_(s1)-casein X-5P (f59-79) (SEQ ID NO: 1, Bos β-casein X-4P (f1-25)(SEQ ID: NO: 2), Bos α_(s2)-casein X-4P (f46-70) (SEQ ID NO: 3) and Bosα_(s2)-casein X-4P (f1-21) (SEQ ID NO: 4) were disclosed in U.S. Pat.5,015,628 as follows:

(SEQ ID NO: 1)Gln⁵⁹-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln-Lys⁷⁹.α_(s1)(59-79)

(SEQ ID NO: 2)Arg¹-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Thr-Arg²⁵.β(1-25)

(SEQ ID NO: 3)Asn⁴⁶-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys⁷⁰.α_(s2)(46-70)

(SEQ ID NO: 4)Lys¹-Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu-Thr-Tyr-Lys²¹.α_(s2)(1-21)

The preliminary determination of the above phosphopeptides for use incombination with CaHPO₄ and hydroxyaptite provided novel peptides havinganticariogenic properties. However, subsequent investigations havedetermined that the Ser(P) cluster sequence motif within the previousdisclosed phosphopeptides have the unexpected ability to stabilize theirown weight in amorphous calcium phosphate. The ability of the abovephosphopeptides and in particular the Ser(P) motif to stabiliseamorphous calcium phosphate was quite unexpected and neither disclosedor taught in any publications known to the Applicants. We have now foundthat the amorphous form of calcium phosphateCa₃(PO₄)_(1.87)(HPO₄)_(0.2)xH₂O where x≧1 stabilised by the caseinphosphopeptides is the most soluble, basic form of non-crystallinecalcium phosphate and a superior form of calcium phosphate whichprevents caries and increases calcium bioavailability. Amorphous calciumphosphate (ACP) must be formed by careful titration of Ca ions (egCaCl₂) and phosphate ions (eg Na HPO₄) while maintaining the pH above 7(preferably 9.0) in the presence of the phosphopeptide. As the ACP isformed, the phosphopeptide binds to the nascent nuclei and stabilisesthe ACP as a phosphopeptide-ACP complex. Without the phosphopeptide, theACP will precipitate out of solution and transform within minutes into,the most stable calcium phosphate phase, crystalline hydroxyapatite(HA). HA, by being insoluble has limited anticariogenic activity andpresents calcium in a poorly bioavailable form. The acidic phase ofcalcium phosphate CaHPO₄, while certainly being more soluble thanhydroxyapatite, is poorly bound by the phosphopeptide and poorlylocalised at the tooth surface and therefore also has limitedanticariogenic activity. The unexpected ability of the aforementionedphosphopeptides and in particular Ser(P) cluster motif to stabilizeamorphous calcium phosphate was not disclosed or taught in U.S. Pat. No.5,015,628 and provides for the first time a reliable and effectivemethod of producing a stabilized amorphous calcium phosphate complexhaving distinct and novel advantages in calcium treatments and delivery.U.S. Pat. No. 5,015,628 does not disclose the unique amorphous calciumfluoride phosphate phase Ca₈(PO₄)₅F x H₂O where x≧1 which we have nowfound to be stabilised by the above phosphopeptides and can be localisedat the tooth surface to provide superior anticaries efficacy. Thisunexpected ability to stabilize amorphous calciun phosphate forms thebasis of the instant invention.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a stable calcium phosphatecomplex, comprising amorphous calcium phosphate or a derivative thereofstabilized by a phosphopeptide, wherein said phosphopeptide comprisesthe sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu-(SEQ ID NO: 5).

In one embodiment, the complex may include phosphopeptide stabilizedamorphous calcium fluoride phosphate.

The phosphopeptide (PP) may be from any source; it may be obtained bytryptic digestion of casein or other phospho-acid rich proteins such asphosphitin, or by chemical or recombinant synthesis, provided that itcomprises the core sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-(SEQ ID NO:5). The sequence flanking this core sequence may be any sequence.However, those flanking sequences in α_(s1)(59-79) (SEQ ID NO: 1),β(1-25) (SEQ ID NO: 2), α_(s2)(46-70 (SEQ ID NO: 3) and α_(s2)(1-21)(SEQ ID NO: 4) are preferred. The flanking sequences may optionally bemodified by deletion, addition or conservative substitution of one ormore residues. The amino acid composition and sequence of the flankingregion are not critical as long as the conformation of the peptide ismaintained and that all phosphoryl and carboxyl groups interacting withcalcium ions are maintained as the preferred flanking regions appear tocontribute to the structural action of the motif.

When the complex takes the form of phosphopeptide stabilized amorphouscalcium fluoride phosphate, the calcium fluoride phosphate may be of theapproximate formula [Ca(PO₄)₅ Fx H₂O] wherein x≧1.

The complex may firer include HPO₄ as a minor optional component to thecomplex. The HP04 is believed to act as a coating for the ACP cluster.When the complex takes the alternative form of a stable soluble alkalinecalcium phosphate complex including stabilized amorphous calciumphosphate, the amorphous calcium phosphate may be of the approximateformula [Ca₃(PO₄)₂ x H₂O] wherein x≧1.

The complex may firther include HPO₄ as a minor optional component. Thecomplex most preferably has a pH of about 9.0.

The phosphopeptide (PP) may be from any source; it may be obtained bytryptic digestion of casein or other phospho-acid rich proteins such asphosphitin, or by chemical or recombinant synthesis, provided that itcomprises the core sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-. The sequenceflanking this core sequence may be any sequence. However, those flankingsequences in α_(s1)(59-79) [1], β(1-25) [2], α_(s2)(46-70) [3] andα_(s2)(1-21) [4] are preferred. The flanking sequences may optionally bemodified by deletion, addition or conservative substitution of one ormore residues. The amino acid composition and sequence of the flankingregion are not critical as long as the conformation of the peptide ismaintained and that all phosphoryl and carboxyl groups interacting withcalcium ions are maintained as the preferred flanking regions appear tocontribute to the structural action of the motif.

The complex formed preferably has the formula [(PP)(CP)₈]_(n) where n isequal to or greater than 1, for example, 6. The complex formed may be acolloidal complex.

The phosphopeptide binds to the ACP cluster to produce a metastablesolution in which growth of ACP to a size that initiates nucleation andprecipitation is prevented. In this way, calcium and other ions such asfluoride ions can be localised, for instance at a surface on a tooth toprevent demineralisation and prevent formation of dental caries.

Thus, in a second aspect, the invention provides a stable calciumphosphate complex as described above, which complex acts as a deliveryvehicle that co-localises ions including, but not limited to calcium,fluoride and phosphate ions at a target site. In a preferred embodiment,the complex is in a slow-release amorphous form that produces superioranti-caries efficacy.

In a particularly preferred embodiment of the invention, the stablecalcium complex is incorporated into dentifrices such as toothpaste,mouth washes or formulations for the mouth to aid in the preventionand/or treatment of dental caries or tooth decay. The calcium complexmay comprise 0.05-50% by weight of the composition, preferably 1.0-50%.For oral compositions, it is preferred that the amount of the CPP-ACPand/or CPP-ACFP administered is 0.05-50% by weight, preferably 1.0%-50%by weight of the composition. The oral composition of this inventionwhich contains the above-mentioned agents may be prepared and used invarious forms applicable to the mouth such as dentifrice includingtoothpastes, toothpowders and liquid dentifrices, mouthwashes, troches,chewing gums, dental pastes, gingival massage creams, gargle tablets,dairy products and other foodstuffs. The oral composition according tothis invention may flirter include additional well known ingredientsdepending on the type and form of a particular oral composition.

In certain highly preferred forms of the invention the oral compositionmay be substantially liquid in character, such as a mouthwash or rinse.In such a preparation the vehicle is typically a water-alcohol mixturedesirably including a humnectant as described below. Generally, theweight ratio of water to alcohol is in the range of from about 1:1 toabout 20:1. The total amount of water-alcohol mixture in this type ofpreparation is typically in the range of from about 70 to about 99.9% byweight of the preparation. The alcohol is typically ethanol orisopropanol. Ethanol is preferred.

The pH of such liquid and other preparations of the invention isgenerally in the range of from about 5 to about 9 and typically fromabout 7.0-9.0. The pH can be controlled with acid (e.g. citric acid orbenzoic acid) or base (e.g. sodium hydroxide) or buffered (as withsodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogenphosphate, sodium dihydrogen phosphate, etc).

In other desirable forms of this invention, the oral composition may besubstantially solid or pasty in character, such as toothpowder, a dentaltablet or a toothpaste (dental cream) or gel dentifrice. The vehicle ofsuch solid or pasty oral preparations generally contains dentallyacceptable polishing material. Examples of polishing materials arewater-insoluble sodium metaphosphate, potassium metaphosphate,tricalcium phosphate, dihydrated calcium phosphate, anhydrous dicalciumphosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesiumphosphate, calcium carbonate, hydrated alumina, calcined alumina,aluminum silicate, zirconium silicate, silica, bentonite, and mixturesthereof. Other suitable polishing material include the particulatethermosetting resins such as melamine-, phenolic, andurea-formaldehydes, and cross-linked polyepoxides and polyesters.Preferred polishing materials include crystalline silica having particlesized of up to about 5 microns, a mean particle size of up to about 1.1microns, and a surface area of up to about 50,000 cm²/gm., silica gel orcolloidal silica, and complex amorphous alkali metal aluminosilicate.

When visually clear gels are employed, a polishing agent of colloidalsilica, such as those sold under the trademark SYLOID as Syloid 72 andSyloid 74 or under the trademark SANTOCEL as Santocel 100, alkali mealalumino-silicate complexes are particularly usefull since they haverefractive indices close to the refractive indices of gellingagent-liquid (including water and/or humectant) systems commonly used indentifrices.

Many of the so-called “water insoluble” polishing materials are anionicin character and also include small amounts of soluble material. Thus,insoluble sodium metaphosphate may be formed in any suitable manner asillustrated by Thorpe's Dictionary of Applied Chemistry, Volume 9, 4thEdition, pp. 510-511. The forms of insoluble sodium metaphosphate knownas Madrell's salt and Kurrol's salt are farther examples of suitablematerials. These metaphosphate salts exhibit only a minute solubility inwater, and therefore are commonly referred to as insolublemetaphosphates (IMP). There is present therein a minor amount of solublephosphate material as impurities, usually a few percent such as up to 4%by weight. The amount of soluble phosphate material, which is believedto include a soluble sodium trimetaphosphate in the case of insolublemetaphosphate, may be reduced or eliminated by washing with water ifdesired The insoluble alkali metal metaphosphate is typically employedin powder form of a particle size such that no more than 1% of thematerial is larger than 37 microns.

The polishing material is generally present in the solid or pastycompositions in weight concentrations of about 10% to about 99%.Preferably, it is present in amounts from about 10% to about 75% intoothpaste, and from about 70%/o to about 99% in toothpowder. Intoothpastes, when the polishing material is silicious in nature, it isgenerally present in amount of about 10-30% by weight, Other polishingmaterials are typically present in amount of about 30-75% by weight.

In a toothpaste, the liquid vehicle may comprise water and humectanttypically in an amount ranging from about 10% to about 80% by weight ofthe preparation. Glycerine, propylene glycol, sorbitol and polypropyleneglycol exemplify suitable humectants/carriers. Also advantageous areliquid mixtures of water, glycerine and sorbitol. In clear gels wherethe refractive index is an important consideration, about 2.5-30% w/w ofwater, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitolare preferably employed.

Toothpaste, creams and gels typically contain a natural or syntheticthickener or gelling agent in proportions of about 0.1 to about 10,preferably about 0.5 to about 5% w/w. A suitable thickener is synthetichectorite, a synthetic colloidal magnesium alkali metal silicate complexclay available for example as Laponite (e.g. CP, SP 2002, D) marketed byLaporte Industries Limited. Laponite D is, approximately by weight58.00% SiO₂, 25.40% MgO, 3.05% Na₂O, 0.98% Li₂O, and some water andtrace metals. Its true specific gravity is 2.53 and it has an apparentbulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gumtragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose,hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose,hydroxyethyl cellulose (e.g. available as Natrosol), sodiumcarboxymethyl cellulose, and colloidal silica such as finely groundSyloid (e.g. 244). Solubilizing agents may also be included such ashumectant polyols such propylene glycol, dipropylene glycol and hexyleneglycol, cellosolves such as methyl cellosolve and ethyl cellosolve,vegetable oils and waxes containing at least about 12 carbons in astraight chain such as olive oil, castor oil and petrolatum and esterssuch as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparationsare to be sold or otherwise distributed in suitable labelled packages.Thus, a jar of mouthrinse will have a label describing it, in substance,as a mouthrinse or. mouthwash and having directions for its use; and atoothpaste, cream or gel will usually be in a collapsible tube,typically aluminium, lined lead or plastic, or other squeeze, pump orpressurized dispenser for metering out the contents, having a labeldescribing it, in substance, as a toothpaste, gel or dental cream.

Organic surface-active agents are used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, nonionic or ampholytic in nature and preferably doesnot interact with the active agent. It is preferred to employ as thesurface-active agent a detersive material which imparts to thecomposition detersive and foaming properties. Suitable examples ofanionic surfactants are water-soluble salts of higher fatty acidmonoglyceride monosulfates, such as the sodium salt of the monosulfatedmonoglyceride of hydrogenated coconut oil fatty acids, higher alkylsulfates such as sodium lauryl sulfite, alkyl aryl sulfonates such assodium dodecyl benzene sulfonate, higher alkylsulfo-acetates, higherfatty acid esters of 1,2-dihydroxy propane sulfonate, and thesubstantially saturated higher aliphatic acyl amides of lower aliphaticamino carboxylic acid compounds, such as those having 12 to 16 carbonsin the fatty acid, alkyl or acyl radicals, and the like. Examples of thelast mentioned amides are N-lauroyl sarcosine, and the sodium,potassium, and ethanolamine salts of N-lauroyl, N-myristoyl, orN-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. The use of these sarconite compoundsin the oral compositions of the present invention is particularlyadvantageous since these materials exhibit a prolonged marked effect inthe inhibition of acid formation in the oral cavity due to carbohydratesbreakdown in addition to exerting some reduction in the solubility oftooth enamel in acid solutions. Examples of water-soluble nonionicsurfactants suitable for use are condensation products of ethylene oxidewith various reactive hydrogen-containing compounds reactive therewithhaving long hydrophobic chains (e.g. aliphatic chains of about 12 to 20carbon atoms), which condensation products (“ethoxamers”) containhydrophilic polyoxyethylene moieties, such as condensation products ofpoly (ethylene oxide) with fatty acids, fatty alcohols, fatty amides,polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide(e.g. Pluronic materials).

The surface active agent is typically present in amount of about 0.1-5%by weight. It is noteworthy, that the surface active agent may assist inthe dissolving of the active agent of the invention and thereby diminishthe amount of solibilizing humectant need.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavouring or sweetening material may also be employed.Examples of suitable flavouring constituents are flavouring oils, e.g.oil of spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavour and sweetening agents may each or together comprisefrom about 0.1% to 5% more of the preparation

In the preferred practice of this invention an oral compositionaccording to this invention such as mouthwash or dentifrice containingthe composition of the present invention is preferably applied regularlyto the gums and teeth, such as every day or every second or third day orpreferably from 1 to 3 times daily, at a pH of about 4.5 to about 9,generally about 7.0 to about 9, for at least 2 weeks up to 8 weeks ormore up to a lifetime.

The compositions of this invention can also be incorporated in lozenges,or in chewing gum or other products, e.g. by stirring into a warm gumbase or coating the outer surface of a gum base, illustrative of whichmay be mentioned jelutong, rubber latex, vinylite resins, etc.,desirably with conventional plasticizers or softeners, sugar or othersweeteners or such as glucose, sorbitol and the like.

In another embodiment, the complex of the invention is formulated toform a dietary supplement preferably comprising 0.1-100% w/w, morepreferably 1-50% w/w, most preferably 1-10% and particularly 2% w/w. Thecomplex may also be incorporated into food products.

Accordingly, in a third sect the invention provides compositionsincluding pharmaceutical compositions comprising the calcium complex asdescribed together with a pharmaceutically-acceptable carrier. Suchcompositions may be selected from the group consisting of dental,anti-cariogenic compositions, therapeutic compositions and dietarysupplements. Dental compositions or therapeutic compositions may be inthe form of a gel, liquid, solid, powder, cream or lozenge. Therapeuticcompositions may also be in the form of tablets or capsules.

In a fourth aspect, there is provided a method of treating or preventingdental caries or tooth decay comprising the step of administering acomplex or composition of the invention to the teeth or gums of asubject in need of such treatments. Topical administration of thecomplex is preferred.

In a fifth aspect, the invention relates to methods of treating one ormore conditions related to calcium loss from the body, especially fromthe bones, calcium deficiency, calcium malabsorption, or the like.Examples of such conditions include, but are not limited to,osteoporosis and osteomalacia. In general any condition which can beimproved by calcium bioavailability is contemplated

In a sixth aspect, the invention also provides a method of producing astable complex of calcium phosphate as described above, comprising thestep of:

(i) obtaining a solution of phosphopeptide having a pH of about 9.0;

(ii) admixing (i) with solutions comprising calcium, and inorganicphosphate and optionally fluoride at a pH of about 9.0;

(iii) filtering the mixture resulting from step (ii), and

(iv) drying to obtain the said complex.

The complexes of the invention are useful as calcium supplements insubjects in need of stimulation of bone growth, for example subjectsundergoing frature repair, joint replacement, bone grafts, orcraniofacial surgery.

These complexes are also useful as dietary supplements m subjects whofor any reason, such as dietary intolerance, allergy, or religious orcultural factors, are unable or unwilling to consume dairy products inan amount sufficient to supply their dietary calcium requirements.

It will be clearly understood tat, although this specification refersspecifically to applications in humans, the invention is also useful forveterinary purposes. Thus in all aspects the invention is useful fordomestic animals such as cattle, sheep, horses and poultry; forcompanion animals such as cats and dogs; and for zoo animals.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference onlyto the following non-limiting Examples.

EXAMPLE 1 Preparation of CPP-ACP and CPP-ACFP

A. Preparation of CPP-ACP

A 10% w/v casein (Murray Goulburn, Victoria, Australia) or caseinatesolution was prepared at pH 8.0 and then digested with trypsin at 0.2%w/w of the casein for 2 h at 50° C. with the pH controlled to 8.0±0.1 byNaOH addition. After digestion the solution was adjusted to pH 4.6 bythe addition of HCl and the precipitate removed by centrifugation ormicrofiltration. However, the solution can also be clarified bymicrofiltration at pH 8.0 without acidification. The supernatant ormicrofiltrate was then adjusted to pH 9.0 with NaOH, then CaCl₂ (1.6 M)and Na₂HPO₄ (1 M) at pH 9.0 were added slowly (≦1% vol per min) withconstant agitation with the pH held constant at 9.0±0.1 by NaOHaddition. CaCl₂ and sodium phosphate were added to the finalconcentrations of 100 mM and 60 mM respectively. Following the additionof the calcium and phosphate solutions, the solution was microfilteredthrough a 0.1 or 0.2 μm microfilter (ceramic or organic) to concentratethe solution five fold. The retentate was then diafiltered with one tofive volumes of distilled water. The retentate after diafiltration wasspray-dried to produce a white powder that was 50% CPP and 40% ACP andresidue water. Analysis of the CPP of the CPP-ACP complex byreversed-phase HPLC, sequence analysis and mass spectrometry revealedthat the only peptides that are capable of stabilizing the amorphouscalcium phosphate and retained during the microfiltration anddiafiltration are Bos α_(s1)-casein X-5P (f59-79) (SEQ ID NO: 1), Bosβ-casein X4P (f1-25) (SEQ ID NO: 2), Box α_(s2)-casein X-4P (f46-70)(SEQ ID NO: 3) and Bos α_(s2)-casein X-4P (f1-21) (SEQ ID NO: 4) andtruncated and heat modified forms of these peptides.

B. Preparation of CPP-ACFP

A 10% w/v casein or caseinate solution was prepared at pH 8.0±0.1 andthen digested with trypsin at 0.2% w/w of the casein for 2 h at 50° C.After digestion the solution was adjusted to pH 4.6 by the addition ofHCl and the precipitate removed by centrifugation or microfiltration.However the solution can also be clarified by microfiltration at pH 8.0without acidification. The supernatant or microfiltrate was thenadjusted to pH 9.0 with NaOH, then CaCl₂ (1.6 M), Na₂HPO₄ (1 M) at pH9.0 and 200 mM NaF were added slowly (≦1% vol per min) with constantagitation with the pH held constant at 9.0±0.1 by NaOH addition. CaCl₂,sodium phosphate and NaF were added to the final concentations of 100mM, 60 mM and 12 mM respectively. Following the addition of the calcium,phosphate and fluoride solutions the solution was microfiltered througha 0.1 or 0.2 μm microfilter (ceramic or organic) to concentrate thesolution five fold. The retentate was then diafiltered with one to fivevolumes of distilled water. The retentate after diafiltration wasspraydried to produce a white powder that was 50% CPP and 40% ACFP andresidue water.

The powdered CPP-ACFP was then reconstituted in distilled water toproduce highly concentrated solutions. For example, a 10% w/v CPP-ACFPsolution containing 640 mM Ca, 400 mM phosphate and 80 mM F (1,520 ppmF) at pH 9.0 has been prepared as well as a 20% CPP gel containing 1.28M Ca, 800 mM phosphate and 160 mM F (3,040 ppm F) at pH 9.0. Thissolution and gel exhibit a significantly grater anticariogenicityrelative to the fluoride alone and therefore are superior additives totoothpaste and mouthwash and for professional application to improve theefficacy of the current fluoride-containing dentifices andprofessionally-applied products.

EXAMPLE 2 Structural Studies of CCP-ACP

A. Structure and Interaction of CCP-ACP

Casein phosphopeptides containing the Ser(P) cluster, i.e. the coresequence motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu-(SEQ ID NO: 5), have amarked ability to stabilize calcium phosphate in solution. Solutionscontaining 0.1% w/v α_(s1)(59-79) (SEQ ID NO: 1) at various pH, calciumand phosphate concentrations, but constant ionic strengths were used tocharacterize the peptide's interaction with calcium phosphate. Thepeptide was found to maximally bind 24 Ca and 16 Pi per molecule asshown in Table 1.

The ion activity products for the various calcium phosphate phases[hydroxyapatite (HA); octacalcium phosphate (OCP); tricalcium phosphate(TCP); amorphous calcium phosphate (ACP); and dicalcium phosphatedihydrate (DCPD) were determined from the free calcium and phosphateconcentrations at each pH using a computer program that calculates theion activity coefficients through the use of the expanded Debye-Hückelequation and takes into account the ion pairs CaHPO₄ ^(o), CaH₂PO₄ ⁺,CaPO₄ ⁻ and CaOH⁺ the dissociation of H₃PO₄ and H₂O and the ionicstrength. The only ion activity product that significantly correlatedwith calcium phosphate bound to the peptide independently of pH was thatcorresponding to ACP [Ca₃(PO₄)_(1.87)(HPO₄)_(.02X)H₂O] indicating thatthis is the phase stabilized by α_(s1)(59-79) SEQ ID NO: 1. The peptideα_(s1)(59-79) (SEQ ID NO: 1) binds to forming ACP clusters producing ametastable solution preventing ACP growth to the critical size requiredfor nucleation and precipitation. The binding of α_(s1)(59-79) (SEQ IDNO: 1) to ACP results in the formation of colloidal complexes with theunit formula [α_(s1)(59-79)(SEQ ID NO: 1)(ACP)₈]_(n) where n is equal toor greater than one. It is likely that the predominant form is n=6 asα_(s1)(59-79) (SEQ ID NO: 1) cross-linked with glutaraldehyde in thepresence of ACP runs as a hexamer on polyacrylamide gel electrophoresis.Interestingly, the synthetic octapeptide α_(s1)(63-70)AcGlu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-GluNHMe (SEQ ID NO: 6) onlybinds 12 Ca and 8 Pi per molecule i.e. (ACP)₄ and the synthetic peptidescorresponding to the N-terminus α_(s1) (59-63), Gln-Met-Glu-Ala-Glu (SEQID NO: 7) and the C-terminus α_(s1)(71-78),Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln (SEQ ID NO: 8) of α_(s1)(59-79) didnot bind calcium phosphate as shown in Table 1. These results indicatethat conformational specificity is essential for full ACP binding.

B. NMR Studies

Protein flexibility in solution is the outstanding characteristic toemerge from spectroscopy studies on proteins containing the Ser(P)cluster sequence (-Ser(P)-Ser(P)-Ser(P)-) such as phosvitin from eggyolk and phosphophoryn from tooth dentine. Phosphorylation appears todestabilise secondary and tertiary structure rather than promote higherlevels of ordering. However, flexible phosphorylated sequences adaptmore regular conformations when bound to calcium phosphate. Opticalrotatory dispersion (ORD), circular dichroism (CD), hydrodynamic and ³¹Pnuclear magnetic resonance (NMR) measurements of the caseins allindicate that α_(s1)-casein and β-casein have a rather open structure insolution with many amino acid side chains exposed to solvent andrelatively flexible. ³¹P-NMR relaxation measurements indicate thatSer(P) residues are relatively mobile in β-casein.

We have demonstrated medium- and long-range nuclear Overhauserenhancements (nOes) in 2D ¹H NMR spectra of α_(s1)(59-79) (SEQ ID NO: 1)in the presence of Ca²⁺ indicating a conformational preference. Twostructured regions were identified. Residues Val72 to Val76 areimplicated in a α-turn conformation. Residues Glu61 to Ser(P)67, whichextend over part of the Ser(P) cluster motif-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- (SEQ ID NO: 5) are involved in aloop-type structure. 2D NMR studies on β-casein(1-25) (SEQ ID NO: 2) inthe presence of calcium have shown a medium range nOe in the-Ser(P)¹⁷-Ser(P)-Ser(P)-Glu-Glu²¹- (SEQ ID NO: 5) motif region betweenthe CαH of Ser(P)¹⁸ and NH of Blu²⁰. Further medium range noes includeone between the CαH of Ser²² and NH of Thr²⁴. Evidence from the ¹H NMRspectra of α_(s2)-casein(1-21) [4] have shown that several residuesincluding those around the -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- (SEQ ID NO: 5)are perturbed. Furthermore, there are medium range nOes between NH ofSer(P)⁸ and NH of GLU¹⁰. This is yet another example of a medium rangenOe in the -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- (SEQ ID NO: 5) motif. Otherexamples of medium range nOes include that between the NH of Ile¹⁴ andNH of Ser(P)¹⁶.

In summary the NMR data indicates that preferred conformations exist forthese peptides in the presence of calcium ions. Molecular modeling ofboth α_(s1)(59-79) (SEQ ID NO: 1) and β(1-25) (SEQ ID NO: 2) using theconstraints derived from the NMR spectroscopy have indicated that thepeptides adopt conformations that allow both glutarnyl and phosphoserylside chains of the cluster motif -Ser(P)-Ser(P)-Ser(P)-Glu-Glu (SEQ IDNO: 5) to interact collectively with calcium ions of the ACP.

The relationship between CPP structure and interaction with amorphouscalcium phosphate was investigated using a series of synthetic peptidehomologues and analogues indicated in Table 1. These studies showed thatthe cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- (SEQ ID NO: 5) wasmainly responsible for the interaction with ACP and that all threecontiguous Ser(P) residues are required for maximal interaction withACP.

TABLE 1 Calcium Phosphate Binding by CPP and Synthetic Homologues andAnalogues V_(ca) V_(Pl) mol/mol mol/mol Ca/P (SEQ ID NO: 5)ΣΣΣEE 9 6 1.5(SEQ ID NO: 9)SΣΣEE 2 1 2.0 (SEQ ID NO: 10)EΣΣEE 2 1 2.0 (SEQ ID NO:11)DΣΣEE 2 1 2.0 (SEQ ID NO: 12)θθθEE 9 6 1.5 (SEQ ID NO: 13)SθθEE 2 12.0 (SEQ ID NO: 14)AΣAE 0 0 (SEQ ID NO: 15)IAΣAEA 0 0 (SEQ ID NO:16)EAIAΣAEA 0 0 (SEQ ID NO: 17)AΣAΣAE 0 0 (SEQ ID NO: 18)AΣAΣAΣAE 2 11.5 (SEQ ID NO: 19)AΣAΣAΣAΣAE 6 4 1.5 (SEQ ID NO: 1)α_(a1) (59-79) 24 161.5 QMEAEΣIΣΣΣEEIVPNΣVEQK (SEQ ID NO: 6)α_(a1) (63-70) EΣIΣΣΣEE 12 8 1.5(SEQ lD NO: 5)α_(a1) (66-70) ΣΣΣEE 9 6 1.5 (SEQ ID NO: 8)α_(a1) (71-78)IVPNΣVEQ 0 0 (SEQ ID NO: 7)α_(a1) (59-63) QMEAE 0 0 (SEQ ID NO: 2)β(1-25) 24 16 1.5 RELEELNVPGEIVEΣLΣΣΣEESITR (SEQ ID NO: 20)β (14-21)EΣLΣΣΣEE 12 8 1.5 Σ = Ser(P), θ = Thr(P), E = Glu, D = Asp, S = Ser, A =Ala, I = Ile, Q = Gln, M = Met, V = Val, P = Pro, K = Lys, L = Leu, T =Thr, G = Gly and R = Arg.

EXAMPLE 3 Structural Studies Using Hydroxyapatite (HA)

Similarly, we investigated the adsorption of the CPP and synthetichomologues and analogues onto HA (Table 2). These data also confirm thatthe Ser(P) cluster sequence is the major determinant for high affinitybinding and that all three coutiguous Ser(P) residues are essential asloss of any one, even when substituted with a Glu or Asp, resulted in aconsiderably lower affinity contant K as shown in Table 2.

TABLE 2 CPP and Synthetic Peptide binding to HA at 37° C. K N Molecularml/μmol μmol/m² Area nm² (SEQ ID NO: 1)α_(a1) (59-79) 415 0.35 4.75QMEAEΣIΣΣΣEEIVPNΣVEQK (SEQ ID NO: 6)α_(a1) (63-70) EΣIΣΣΣEE 10,370 0.473.56 (SEQ ID NO: 5)α_(a1) (66-70) ΣΣΣEEE 12,845 0.52 3.27 (SEQ ID NO:8)α_(a1) (71-78) — — — IVPNΣVEQ (SEQ ID NO: 7)α_(a1) (59-63) QMEAE — — —(SEQ ID NO: 5)ΣΣΣEE 12,845 0.52 3.27 (SEQ ID NO: 10)EΣΣEE 1,513 0.961.74 (SEQ ID NO: 11)DΣΣEE 6,579 0.81 2.04 (SEQ ID NO: 12)θθθEE 12,2340.51 3.27 (SEQ ID NO: 21)TθθEE 1,013 0.55 3.03 (SEQ ID NO: 22)θTθEE 8370.44 3.77 (SEQ ID NO: 23)θθTEE 1,799 0.46 3.61 Σ = Ser(P), θ = Thr(P)

Interestngly, repeating these HA adsorption experiments with salivarycoated HA (sHA) revealed that the Ser(P) cluster motif was still themajor determinant for adsorption although the affinities of the peptidesfor the sHA was slightly reduced by the presence of the salivaryproteins. These results suggest that the predominant interaction of theCPP with pellicle and plaque is likely to be electrostatic and mediatedby the Ser(P) cluster motif of the CPP.

We have also studied the docking of the peptide Ser(P)- Ser(P)-Ser(P)-Glu-Glu- onto three crystallographic planes of HA, {100}, {010}and {001} using computer simulation techniques and the unit cellcoordinates of synthetic HA. These simulation studies revealed that thepeptide -Ser(P)- Ser(P)- Ser(P)-Glu-Glu- is more likely to the {100}surface, followed by the {010} surface. The Ser(P)- cluster motif cantherefore bind to both {100} and {010} surfaces thus allowing depositionof calcium, phosphate and hydroxyl ions on the {100} surface enablinggrowth of the HA crystal along the c-axis only. These results thereforecan know explain the c-axis growth of HA crystals in enamel and dentine.Detailed examination of the computer simulation data shows that the-Ser(P)- Ser(P)- Ser(P)-Glu-Glu- conformer with the greatest relativebinding energy is positioned on the HA surface such that the carboxylgroups of the glutamyl residues and the phosphoryl groups of thephosphoseryl residues are in proximity to the HA surface with maximalcontact between these groups and surface calcium atoms.

EXAMPLE 4 Anticariogenic Activity of CPP-ACP in Human in situ Studies

The ability of the 1.0% w/v CPP-ACP pH 7.0 solution to prevent enameldemineralisation was studied in a human in situ caries model. The modelconsists of a removable appliance containing a left and right pair ofenamel slabs placed to produce a plaque retention site. The inter-enamelplaque that developed (3-5 mg) was bacteriologically similar to normalsupragingival plaque. On frequent exposure to sucrose solutions over athree week period, the increase in levels of mutans streptococci andlactobacilli and in sub-surface enamel demineralisation resulted in theformation of incipient “caries-like” lesions.

Two exposures of the CPP-ACP solution per day to the right pair ofenamel slabs for 12 subjects produced a 51%±19% reduction in enamelmineral loss relative to the left-side, control enamel. The plaqueexposed to the CPP-ACP solution contained 78±22 μmol/g calcium, 52±25μmol/g P₁ and 2.4±0.7 mg/g CPP compared with 32±12 μmol/g calcium and20±11 μmol/g P₁ in the control plaque. The level of the CPP wasdetermined by competitive ELISA using an antibody that recognizes bothα_(s1)(59-79) and β(1-25). Electron micrographs of immunocytochemicallystained sections of the plaque revealed localization of the peptidepredominantly on the surface of microorganisms but also in theextracellular matrix.

Although these results indicate that CPP are incorporated intodeveloping dental plaque, the actual level determined by ELISA would notbe a true representation of that incorporated due to the breakdown ofthe CPP in plaque through the action of phosphatase and peptidaseactivities. The incorporation of the CPP-ACP in the plaque resulted in a2.4 fold increase in the plaque calcium and a 2.6 fold increase inplaque P₁ with a Ca/P₁ ratio consistent with ACP.

EXAMPLE 5 Anticariogenic Potential of the CPP-ACP In a Mouthwash Study

A clinical trial of a mouthwash used thrice daily containing 3.0%CPP-ACP pH 9.0 showed that the calcium content of supragingival plaque(lower anterior teeth excluded) increased from 169±103 μmol/g dry weightto 610±234 μmol/g after use of the mouthwash for a three day period, andinorganic phosphate increased from 242±60 μmol/g dry weight to 551±164μmol/g. These post-mouthwash levels of calcium and inorganic phosphateare the highest ever reported for non-mineralised supragingival plaque.

Without wishing to be bound by any proposed mechanism for the observedadvantages, it is believed that the mechanism of anticariogenicity forthe CPP-ACP is the incorporation of amorphous calcium phosphate inplaque, thereby depressing enamel demineralisation and enhancingremineralisation. In plaque, CPP-ACP would act as a reservoir of calciumand phosphate, buffering the free calcium and phosphate ion activitiesthereby helping to maintain a state of supersaturation with respect totooth enamel. The binding of ACP to CPP is pH dependent with very littlebound below pH 7.0.

EXAMPLE 6 Remineralisation of Enamel Lesions by CPP-ACP

A. In Vitro Studies

An in vitro enamel remineralisation system was used to studyremineralisation of artificial lesions in human third molars by CPP-ACPsolutions. Using this system, a 1.0% CPP-ACP solution replaced 56±21% ofmineral lost. A 0.1% CPP-ACP solution replaced 34±18% of mineral lost. Afurther number of solutions containing various amounts of CPP(0.1-1.0%), calcium (6-60 mM) and phosphate (3.6-36 mM) at different pHvalues (7.0-9.0) were prepared. The associations between the activitiesof the various calcium phosphate species in solution and the rate ofenamel lesion remineralisation for this series of solutions were thendetermined.

The activity of the neutral ion species CaHO₄ ^(o) in the variousremineralising solutions was found to be highly correlated with the rateof lesion remineralisation. The diffusion coefficient for theremineralisation process was estimated at 3×10⁻¹⁰ m²s⁻¹which isconsistent with the coefficients of diffision for neutral moleculesthrough a charged matrix. The rate of enamel remineralisation obtainedwith the 1.0% CPP-ACP solution was 3.3×10⁻² mol HA/m²/10 days which isthe highest remineralisation rate ever obtained. Calcium phosphate ions,in particular the neutral ion pa CaHPO₄ ^(o), after diffusion into theenamel lesion, will dissociate and thereby increase the degree ofsaturation with respect to HA. The formation of HA in the lesion willlead to the generation of H₃PO₄, which being neutral itself, willdiffuse out of the lesion down a concentration gradient.

The results indicate that the CPP-bound ACP,CPP[Ca₃(PO₄)_(1.87)(HPO₄)_(0.2)xH₂O]₈ acts as a reservoir of the neutralion species, CaHPO₄ ^(o) that is formed in the presence of acid. Theacid can be generated by dental plaque bacteria; under these conditions,the CPP-bound ACP would buffer plaque pH and produce calcium andphosphate ions, in particular CaHPO₄ ^(o). The increase in plaque CaHPO₄^(o) would offset any fall in pH thereby preventing enameldemineralisation Acid is also generated in plaque as H₃PO₄ by theformation of HA in the enamel lesion during remineralisation. Thistherefore explains why the CPP-ACP solutions are such efficientremineralising solutions as they would consume the H₃PO₄ produced duringenamel lesion remineralisation generating more CaHPO₄ ^(o) thusmaintaining its concentration gradient into the lesion. These resultsare therefore consistent with the proposed anticariogenic mechanism ofthe CPP being the inhibition of enamel demineralisation and enhancementof remineralisation through the localisation of ACP at the toothsurface.

B. Human in Situ Remineralisation Studies

The ability of CPP-ACP added to sugar-free (sorbitol) chewing gum toremineralise enamel sub-surface lesions was investigated in arandomized, cross-over, double-blind study. Ten subjects wore removablepalatal appliances with six, human-enamel, half-slabs inset containingsub-race deminealised lesions. The other half of each enamel slab wasstored in a humidified container and was used as the controldeminealised lesion. There were four treatment groups in the study,sugar-free gum containing 3.0% w/w CPP-ACP, sugar-free gum containing1.0% w/w CPP-ACP, sugar-free gum with no CPP-ACP and a no-gum-chewingcontrol. The gums were chewed for 20 min periods, four times a day. Theappliances were worn for this 20 min period and a further 20 min periodafter gum chewing. Each treatment was for 14 days duration and each ofthe ten subjects carried out each treatment with a one week rest betweenthe treatments. At the completion of each treatment the enamel slabswere removed, paired with their respective deminealised control,embedded, sectioned and subjected to microradiography andcomputer-assisted densitometric image analysis to determine the level ofremineralisation. The sugar-free gum treatment resulted in 9.82±1.81%remineralisation relative to the no-gum-chewing control whereas the gumcontaining 1.0% CPP-ACP produced 17.06±2.48% remineralisation and the3.0% CPP-ACP gum produced 22.70±3.40% reminerlisation with all valuesbeing significantly different. These results showed that addition of1.0% and 3.0% CPP-ACP to sugar-free gum produced a 74% and 131% increaserespectively in sub-surface enamel remineralisation.

EXAMPLE 7 CPP-ACFP Mouthwash Study

A mouthwash study was conducted to determine the ability of a 3.0%CPP-ACFP mouthwash used thrice daily to increase supragingival plaquecalcium, inorganic phosphate and fluoride ions. The 3.0% CPP-ACFPsolution used as a mouthwash for four days contained 192 mM boundcalcium ions, 120 mM bound phosphate ions and 24 mM (456 ppm) bound Fions stabilised by CPP. The use of the mouthwash resulted in a 1.9 foldincrease in plaque calcium, a 1.5 fold increase in plaque phosphate anda dramatic 18 fold increase in plaque fluoride ion as shown in Table 3.

TABLE 3 Effect of CPP-ACFP on Plaque, Ca, P_(i) and F Levels Ca Pi Fμmol/g μmol/g μmol/g Control 177 ± 53 306 ± 82  1.1 ± 0.9 3% CPP-ACFP336 ± 107 471 ± 113 19.9 ± 14.1 1000 ppm F 158 ± 54 287 ± 29  1.9 ± 1.03% CaCPP 193 ± 56 343 ± 102  1.5 ± 0.8

Although these marked increases in plaque calcium, phosphate andfluoride were found, dental calculus was not observed in any of thesubjects, suggesting that the plaque calcium fluoride phosphate remainedstabilised as the amorphous phase by the CPP and did not transform intoa crystalline phase. These increases in the supragingival plaque levelsof Ca, phosphate and fluoride ions produced by CPP-ACP are markedlygreater than those obtained in a similar study using CaCPP and 1000 ppmF (MFP and NaF) toothpastes twice daily for a similar time period asindicated in Table 3. These results show a marked synergistic effectbetween fluoride ions and the CPP-ACP. This is particularly advantageousin view of the fact that the level of fluoride in oral compositions suchas toothpaste can then be reduced, resulting in cost savings and loweredrisk of fluorosis for individuals living in high-fluoride areas.

EXAMPLE 8 Interaction of CPP-ACP With Fluoride

An synergistic anticariogenic effect of the 1.0% CPP-ACP together with500 ppm F³¹ was observed in a rat caries model. Analysis of the solutioncontaining 1.0% CPP, 60 mM CaCl₂, 36 mM sodium phosphate and 500 ppm F(26.3 mM NaF) pH 7.0 after ultrafiltration revealed that nearly half ofthe fluoride ion had incorporated into the ACP phase stabilised by theCPP to produce an amorphous calcium fluoride phosphate phase ofcomposition Ca₈(PO₄)₅F xH₂O, with 24 Ca, 15 PO₄ and 3F molecules per CPPmolecule.

Without wishing to be limited by any proposed mechanism for the observedbeneficial effect, we consider that the anticariogenic mechanism of theCPP-ACP is the localisation of ACP at the tooth surface such that in thepresence of acid, the ACP dissociates to release Ca and phosphate ionsincreasing the degree of saturation with sect to HA preventing enameldemineralisation and promoting remineralisation. The anticariogenicmechanism of fluoride is the localisation of the fluoride ion at thetooth surface, particularly in plaque in the presence of Ca andphosphate ions. This localisation increases the degree of saturationwith respect to fluorapatite (FA) thus promoting remineralisation ofenamel with FA. It is clear that for the formation of FA [Ca₁₀(PO₄)₆F₂],calcium and phosphate ions must be co-localised in plaque at the toothsurface with the fluoride ion. The synergistic anticariogenic effect ofCPP-ACP and F is therefore attributable to the localisation of ACFP atthe tooth surface by the CPP which in effect would co-localise Ca, Piand F.

This was demonstrated in the mouthwash study described in Example 7.

Metastable solutions of the CPP at pH 7.0 have been prepared containingamorphous calcium fluoride phosphate at remakably high concentrations.For example, a 10% w/v CPP -ACFP solution containing 640 mM Ca, 400 mMphosphate and 80 mM F (1,520 ppm F) at pH 7.0 has been prepared as wellas a 20% CPP gel containing 1.28 M Ca, 800 mM phosphate and 160 mM F(3,040 ppm F⁻) at pH 7.0. This solution and gel exhibit a significantlygreater anticariogenicity relative to the fluoride alone, and thereforeare superior additives to toothpastes and mouthwash and for professionalapplication to improve the efficacy of the current fluoride-containingdentifrices and professionally-applied products.

Specific examples of formulations containing the complexes of theinvention are provided below.

EXAMPLE 9 Toothpaste Formulations Containing CPP-ACFP

Formulation 1

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Glycerol 20.0 Sodiumcarboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroylsarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1 Chlorhexidine gluconate0.01 Dextranase 0.01 CPP-ACFP 1.00 Water balance

Formulation 2

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase0.01 CPP-ACFP 2.0 Water balance

Formulation 3

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Lauroyl diethanolamide1.0 Sucrose monolaurate 2.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01CPP-ACFP 5.0 Water balance

Formulation 4

Ingredient % w/w Sorbitol 22.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0Gantrez 19.0 Water (deionised) 2.69 Sodium Monofluorophosphate 0.76Sodium saccharine 0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavouroil 0.95 CPP-ACFP 1.0 sodium lauryl sulphate 2.00

Formulation 5

Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3Chlorhexidine gluconate 0.01 Ethanol 3.0 CPP-ACFP 2.0 Linolic acid 0.05Water balance

EXAMPLE 10 Mouthwash Formulations

Formulation 1

Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.3 CPP-ACFP 2.0 Water balance

Formulation 2

Ingredient % w/w Gantrez S-97 2.5 Glycerine 10.0 Flavour oil 0.4 Sodiummonofluorophosphate 0.05 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.2 CPP-ACFP 2.0 Water Balance

EXAMPLE 11 Lozenge Formulation

Ingredient % w/w Sugar 75-80 Corn syrup  1-20 Flavour oil 1-2 NaF0.01-0.05 CPP-ACFP 3.0 Mg stearate 1-5 Water balance

EXAMPLE 12 Gingival Massage Cream Formulation

Ingredient % w/w White petrolatum 8.0 Propylene glycol 4.0 Stearylalcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0Sucrose monostearate 0.5 Chlorohexidine gluconate 0.1 CPP-ACFP 3.0 Waterbalance

EXAMPLE 13 Chewing Gum Formulation

Ingredient % w/w Gum base 30.0 Calcium carbonate 2.0 Crystallinesorbitol 53.0 Glycerine 0.5 Flavour oil 0.1 CPP-ACFP 2.0 Water balance

EXAMPLE 14 Dietary Supplement

CPP-ACP was added at 1.0% w/w of the diet of rachitic chickens todetermine the ability of the CPP-ACP to provide bioavailable calcium forbone accretion. CPP-ACP at 1.0% w/w in the diet produced a 34% reductionin the incidence of growth plate abnormalities, a 17% increase in tibialash and a 22% reduction in the cartilaginous growth plate in the animalswhich was significantly greater than the CPP alone (Table 4) indicatingthat the CPP-ACP is superior to the CPP in providing bioavailabledietary calcium and in facilitating bone accretion.

TABLE 4 Effect of 1.0% CPP-ACP addition to the diet of rachitic chickenson incidence of growth plate abnormalities, tibial ash and cartilaginousgrowth plate width % Growth Growth Plate Abnormalities % % Tibial Ash %Width (mm) Control 53 ± 5 30 ± 2 5.4 ± 0.2 1.0% CPP 47 ± 9 30 ± 2 5.3 ±0.2 1.0% CPP-ACP 35 ± 3 35 ± 1 4.2 ± 0.2

It should be understood that while the invention has been described indetail for the purposes of clarity and understanding, the examples werefor illustrative purposes only. Other modifications of the embodimentsof the present invention will be apparent to those skilled in the art ofmolecular biology, dental diagnostics, and related disciplines and arewithin the scope of the invention as describe

23 1 21 PRT Bos sp. misc_feature (6)..(6) Xaa is a phosphorylated Serine1 Gln Met Glu Ala Glu Xaa Ile Xaa Xaa Xaa Glu Glu Ile Val Pro Asn 1 5 1015 Xaa Val Glu Gln Lys 20 2 25 PRT Bos sp. misc_feature (15)..(15) Xaais a phosphorylated Serine 2 Arg Glu Leu Glu Glu Leu Asn Val Pro Gly GluIle Val Glu Xaa Leu 1 5 10 15 Xaa Xaa Xaa Glu Glu Ser Ile Thr Arg 20 253 25 PRT Bos sp. misc_feature (11)..(11) Xaa is a phosphorylated Serine3 Asn Ala Asn Glu Glu Glu Tyr Ser Ile Gly Xaa Xaa Xaa Glu Glu Xaa 1 5 1015 Ala Glu Val Ala Thr Glu Glu Val Lys 20 25 4 21 PRT Bos sp.misc_feature (8)..(8) Xaa is a phosphorylated Serine 4 Lys Asn Thr MetGlu His Val Xaa Xaa Xaa Glu Glu Ser Ile Ile Xaa 1 5 10 15 Gln Glu ThrTyr Lys 20 5 5 PRT Artificial Sequence misc_feature ()..() Syntheticpeptide 5 Xaa Xaa Xaa Glu Glu 1 5 6 8 PRT Artificial Sequencemisc_feature ()..() Synthetic peptide 6 Glu Xaa Ile Xaa Xaa Xaa Glu Glu1 5 7 5 PRT Artificial Sequence misc_feature ()..() Synthetic peptide 7Gln Met Glu Ala Glu 1 5 8 8 PRT Artificial Sequence misc_feature ()..()Synthetic peptide 8 Ile Val Pro Asn Xaa Val Glu Gln 1 5 9 5 PRTArtificial Sequence misc_feature ()..() Synthetic peptide 9 Ser Xaa XaaGlu Glu 1 5 10 5 PRT Artificial Sequence misc_feature ()..() Syntheticpeptide 10 Glu Xaa Xaa Glu Glu 1 5 11 5 PRT Artificial Sequencemisc_feature ()..() Synthetic peptide 11 Asp Xaa Xaa Glu Glu 1 5 12 5PRT Artificial Sequence misc_feature ()..() Synthetic peptide 12 Xaa XaaXaa Glu Glu 1 5 13 5 PRT Artificial Sequence misc_feature ()..()Synthetic peptide 13 Ser Xaa Xaa Glu Glu 1 5 14 4 PRT ArtificialSequence misc_feature ()..() Synthetic peptide 14 Ala Xaa Ala Glu 1 15 6PRT Artificial Sequence misc_feature ()..() Synthetic peptide 15 Ile AlaXaa Ala Glu Ala 1 5 16 8 PRT Artificial Sequence misc_feature ()..()Synthetic peptide 16 Glu Ala Ile Ala Xaa Ala Glu Ala 1 5 17 6 PRTArtificial Sequence misc_feature ()..() Synthetic peptide 17 Ala Xaa AlaXaa Ala Glu 1 5 18 8 PRT Artificial Sequence misc_feature ()..()Synthetic peptide 18 Ala Xaa Ala Xaa Ala Xaa Ala Glu 1 5 19 10 PRTArtificial Sequence misc_feature ()..() Synthetic peptide 19 Ala Xaa AlaXaa Ala Xaa Ala Xaa Ala Glu 1 5 10 20 8 PRT Artificial Sequencemisc_feature ()..() Synthetic peptide 20 Glu Xaa Leu Xaa Xaa Xaa Glu Glu1 5 21 5 PRT Artificial Sequence misc_feature ()..() Synthetic peptide21 Thr Xaa Xaa Glu Glu 1 5 22 5 PRT Artificial Sequence misc_feature()..() Synthetic peptide 22 Xaa Thr Xaa Glu Glu 1 5 23 5 PRT ArtificialSequence misc_feature ()..() Synthetic peptide 23 Xaa Xaa Thr Glu Glu 15

What is claimed is:
 1. A stable, soluble calcium phosphate complex thatis obtainable by a process comprising the steps of: (i) obtaining ansolution of a phosphopeptide, wherein the phosphopeptide includes aminoacid sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu (SEQ ID NO: 5); (ii)admixing, under alkaline conditions, the solution of step (i) withcalcium ions and inorganic phosphate, to obtain a mixture; and (iii)isolating the complex from the mixture of step (ii).
 2. The complex ofclaim 1, wherein the alkaline conditions are pH of about
 9. 3. Thecomplex of claim 1, wherein the amino acid sequence is selected from thegroup consisting of: (SEQ ID NO: 1)Gln⁵⁹-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln-Lys⁷⁹α_(s1)(59-79); (SEQ ID NO: 2)Arg¹-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Thr-Arg²⁵β(1-25); (SEQ ID NO: 3)Asn⁴⁶-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys⁷⁰α_(s2)(46-70); and (SEQ ID NO: 4)Lys¹-Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu-Thr-Tyr-Lys²¹α_(s2)(1-21).
 4. The complex of claim 3, wherein the amino acid sequenceis: (SEQ ID NO: 1)Gln⁵⁹-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln-Lys⁷⁹α_(s1)(59-79).
 5. A stable, soluble calcium fluoride phosphate complexthat is obtainable by a process comprising the steps of: (i) obtaining asolution of a phosphopeptide, wherein the phosphopeptide includes aminoacid sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu (SEQ ID NO: 5); (ii)admixing, under alkaline conditions, the solution of step (i) withcalcium ions, inorgainc phosphate and fluoride ions, to obtain amixture; and (iii) isolating the complex form the mixture of step (ii).6. The complex of claim 5, wherein the alkaline conditions are pH ofabout
 9. 7. The complex of claim 5, wherein the amino acid sequence isselected from the group consisting of: (SEQ ID NO: 1)Gln⁵⁹-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln-Lys⁷⁹α_(s1)(59-79); (SEQ ID NO: 2)Arg¹-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Thr-Arg²⁵β(1-25); (SEQ ID NO: 3)Asn⁴⁶-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys⁷⁰α_(s2)(46-70); and (SEQ ID NO: 4)Lys¹-Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu-Thr-Tyr-Lys²¹α_(s2)(1-21).
 8. The complex of claim 7, wherein the amino acid sequenceis: (SEQ ID NO: 1):Gln⁵⁹-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln-Lys⁷⁹α_(s1)(59-79).
 9. A method of preparing a stable, soluble calciumphosphate complex comprising the steps of: (i) obtaining a solution of aphosphopeptide, wherein the phosphopeptide includes amino acid sequenceSer(P)-Ser(P)-Ser(P)-Glu-Glu (SEQ ID NO: 5); (ii) admixing the solutionof step (i) with calcium ions and inorganic phosphate at a pH of above7, to obtain a mixture; (iii) filtering the mixture of step (ii) toproduce a retentate, and drying the retentate to isolate the complex.10. The method of claim 9, wherein step (ii) further comprises admixingthe solution of step (i) with fluoride ions in addition to the calciumions and inorganic phosphate.
 11. A stable, soluble calcium phosphatecomplex that is obtainable by the process of claim
 9. 12. A stable,soluble calcium phosphate complex that is obtainable by the process ofclaim
 10. 13. A delivery vehicle for administering a complex of claim 1,5, 11 or 12 to a target site, wherein the delivery vehicle is adapted toco-localize calcium and phosphate ions of the complex at the targetsite.
 14. The delivery vehicle of claim 13 that is selected from thegroup consisting of toothpaste, toothpowder, liquid dentifrice,mouthwash, troche, chewing gum, dental paste, gingival massage cream,gargle tablet and foodstuff.
 15. The delivery vehicle of claim 14,wherein the foodstuff is a dairy product.
 16. A method of treating orpreventing dental caries or tooth decay, comprising administering acomplex according to claim 1, 5, 11 or 12 to the teeth or gums of asubject in need of such treatment.
 17. A pharmaceutical compositioncomprising: (i) a complex of claim 1, 6; and (ii) a pharmaceuticalcarrier or a delivery vehicle.
 18. The pharmaceutical composition ofclaim 17, wherein the complex is present in effective amount forremineralising teeth or for inhibiting cariogenesis or tooth decay in asubject.
 19. The pharmaceutical composition of claim 17, wherein thecomplex is present in an effective amount for promoting calciumabsorption in a subject suffering from a condition related to calciumloss, calcium deficiency or calcium malabsorption.
 20. Thepharmaceutical composition of claim 19, wherein the condition isosteoporosis or osteomalacia.
 21. The pharmaceutical composition ofclaim 17, further comprising foodstuff.
 22. The pharmaceuticalcomposition of claim 21, wherein the foodstuff is a dairy product. 23.The pharmaceutical composition of claim 17, further comprising adentifrice.
 24. The pharmaceutical composition of claim 23, wherein thedentifrice is selected from the group consisting of toothpaste,toothpowder, liquid dentifrice, mouthwash, troche, chewing gum, dentalpaste, gingival massage cream and gargle tablet.
 25. A method forremineralising teeth or for inhibiting cariogenesis or tooth decay,comprising administering an effective amount of a complex of claim 1, 5,11 or 12 to a subject in need thereof.
 26. A method for promotingcalcium absorption in a subject suffering from a condition related tocalcium loss, calcium deficiency or calcium malabsorption, comprisingadministering an effective amount of a complex of claim 1, 5, 11 or 12to the subject.
 27. The method of claim 26, wherein the condition isosteoporosis or osteomalacia.